This project is an effort to uncover the mechanism of post-translational regulation of the pentose phosphate pathway in human red blood cells. A macromolecular factor has been found that seems to represent a new mechanism for intracellular control of metabolism: regulation by cofactor binding. Much of the NADP+ and NADPH of normal red cells is bound by this factor. It has the properties of a protein with an equal number of NADP+ binding sites and NADPH binding sites, each site having lesser affinity for the opposite dinucleotide. This causes the NADP+/NADP ratio of the unbound nucleotide to be exaggerated relative to the corresponding ratio for total (bound and unbound) nucleotide. As a result, a sigmoid curve is obtained when the observed NADP+/NADP ratio of the free nucleotides is plotted against various observed ratios of total nucleotide in concentrated hemolysates. This finding now accounts for the unexplained intracellular restraint and sigmoid kinetics of red cell glucose-6-phosphate dehydrogenase (G6PD or D-glucose-6-phosphate:NADP+ 1-oxidoreductase; E.C. 1.1.1.49), the enzyme which represents the initial and committed step into the pentose phosphate pathway. The factor will be purified and further characterized. Its relationship to the FX protein of DeFlora and to the Eggleston-Krebs factor will be determined, and its role in some of the unexpectedly severe manifestations of G6PD deficiency will be explored. A major thrust in this laboratory is the use of techniques, such as lysis and resealing of red cells and determinations of NADP+ and NADPH concentrations, that might allow: (1) recognition of the major NADPH-regulating factors in the whole cell and lysate, (2) isolation of the factor, and (3) confirmation that the purified factor is probably the counterpart of the predominant intracellular factor.